PROJECT SUMMARY Globally over 2 million children live with HIV and 400 are newly infected every day. There is an urgent need for an HIV cure and one barrier to that is immune exhaustion. The goal of our study is to determine the role of immune exhaustion in pediatric HIV pathogenesis in order to guide innovative approaches toward a functional cure. Exhausted T cells are defined by progressive loss of proliferative, cytotoxic, and cytokine immune responses mediated by the sustained upregulation of multiple inhibitory receptors (IR). Our prior research in a cohort of Kenyan children with perinatal HIV infection demonstrates that HIV+ children have high levels of inhibitory receptor PD-1 on T cells that correlate with HIV disease progression. Moreover, high PD-1 levels predicted lower HIV-specific proliferative responses suggesting that high PD-1 levels may weaken HIV eradication or functional cure strategies. There are several other IRs ? including CD160, lymphocyte activation gene 3 (LAG3), cytotoxic T lymphocyte antigen 4 (CTLA-4), T-cell immunoglobulin domain and mucin domain- containing protein 3 (TIM3), and T-cell immunoreceptor and Ig and ITIM domains (TIGIT) that also dampen immune responses in HIV+ adults. Simultaneous upregulation of multiple IRs links to increasing degrees of immune dysfunction, yet the levels of IRs on T cells and their functional consequences in children with perinatal HIV infection is unknown. The objective of this study is to perform a comprehensive analysis of immune checkpoints including their phenotypic expression, transcriptome, and functional potential in HIV+ children. We will utilize a biobank of peripheral blood mononuclear cells from the Pediatric Immune Activation Study led by Dr. Khaitan that includes 160 children with perinatal HIV infection and 82 HIV-unexposed uninfected controls between ages 2 months to 20 years for this study. We hypothesize there is a unique signature of multiple IR coexpressions on CD4 and CD8 T cells in children with perinatal HIV infection that identifies truly exhausted T cells. Our specific aims are to (1) Determine the phenotype and transcriptome of IR-expressing T cells in children with perinatal HIV infection; and (2) Determine the functional consequences of IRs on CD4 and CD8 T cells and potential for reversal in perinatal HIV infection. In Aim 1 we will apply innovative technologies of 30-parameter flow cytometry and single cell RNA genomic sequencing to extract detailed immunologic and transcriptomic data from limited PBMC numbers obtained from children. In Aim 2 we will determine ex vivo proliferative, cytotoxic, and cytokine responses to HIV peptides and childhood vaccines and in vitro responses to IR inhibitors. Our research will fill fundamental knowledge gaps in understanding the role of immune exhaustion in pediatric HIV pathogenesis, with potential to identify novel targets for future HIV functional cure strategies in children.